Abstract
Stable reference genes are necessary to analyse quantitative real-time reverse transcription PCR (qRT-PCR) data and determine the reliability of the final results. For further studies of the valuable fungus Shiraia bambusicola, the identification of suitable reference genes has become increasingly urgent. In this study, three conventional reference genes and nine novel candidates were evaluated under different light conditions (all-dark, all-light and 12-h light/dark) and in different media (rice medium, PD medium, and Czapek–Dox medium). Three popular software programs (geNorm, NormFinder and BestKeeper) were used to analyse these genes, and the final ranking was determined using RefFinder. SbLAlv9, SbJsn1, SbSAS1 and SbVAC55 displayed the best stability among the genes, while SbFYVE and SbPKI showed the worst. These emerging genes exhibited significantly better properties than the three existing genes under almost all conditions. Furthermore, the most reliable reference genes were identified separately under different nutrient and light conditions, which would help accessible to make the most of the existing data. In summary, a group of novel housekeeping genes from S. bambusicola with more stable properties than before was explored, and these results could also provide a practical approach for other filamentous fungi.
Highlights
IntroductionShiraia bambusicola is an important and valuable macrofungus in the medical and food industries
Shiraia bambusicola is an important and valuable macrofungus in the medical and food industries. It is noteworthy for its hypocrellins, the main secondary metabolites of S. bambusicola, whose use has been proposed for disease treatments that involve anti-clinical strains and antiinflammatory and anti-viral activity (Su et al 2011; Jiang et al 2011; Zhou et al 2009)
Fungal isolates adhered to agars were cultured in potato dextrose agar (PDA) medium at 26 °C for 7 days, and the fresh mycelia were inoculated onto different media under three different light conditions, namely, 24 h of continuous darkness, 24 h of continuous lighting with a light intensity of 1500 lx, and 12: a 12 h light photoperiod with 1500 lx light intensity (12-h light/dark)
Summary
Shiraia bambusicola is an important and valuable macrofungus in the medical and food industries. It is noteworthy for its hypocrellins, the main secondary metabolites of S. bambusicola, whose use has been proposed for disease treatments that involve anti-clinical strains and antiinflammatory and anti-viral activity (Su et al 2011; Jiang et al 2011; Zhou et al 2009). A large number of research papers have been published on reference genes under different stresses or from different organs in plants (Warzybok and Migocka 2013; Lin et al 2014), and similar works in filamentous fungi are gradually being conducted (Zampieri et al 2014; Zhou et al 2011). The classical reference genes elongation factor (EF-1) and beta-tubulin (β-tubulin), which are widely used in plants, were not appropriate in Hemileia vastatrix (Vieira et al 2011); another reliable
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